Alpha-1-acid glycoprotein (AAG) is a plasma glycoprotein of approximate molecular weight 41 kD. It is an acute phase protein, present in plasma at a concentration of between 0.5-1 g/l in healthy people, rising in disease states, particularly inflammatory diseases, to levels up to about 2 g/l.
The physiological role of AAG is poorly understood. As an acute phase protein, its serum level increases in response to a number of stresses and insults including infection, trauma, burns, etc. AAG is known to act on a wide variety of cells and it has been suggested that AAG may play a role in the immune response. In addition, AAG has been shown to bind to a diversity of drugs, particularly basic and lipophilic drugs. Therapeutic uses of AAG based on this latter aspect have been suggested in the literature but none have been actually developed as far as the clinic.
We believe that one reason for this is the relatively high level of contaminants which remain even in so-called highly purified preparations. The endotoxin lipopolysaccharide (LPS) derived from bacterial cell walls, also known as pyrogen, is one such contaminant.
LPS is the causative agent of septic shock, which is a major cause of morbidity following gram-negative bacterial infection, particularly in hospitalised and immunocompromised patients. The presence of LPS in AAG preparations renders them unsuitable for human therapy.
Currently available methods of purifying AAG are laborious and time consuming, involving a large number of individual steps. Furthermore, they are unsuitable for large scale preparative processes. One such technique is described by Hao and Wickerhauser (Biochem. Biophys. Acta, 322, 99-108 (1973)). This involves adsorption and elution of a Cohn Fraction V supernatant from DEAE Sephadex, concentration, dialysis, adsorption and elution from carboxymethylcellulose, dialysis and finally freeze drying. With both dialysis steps taking 48 hours each, the whole process takes over a week. Furthermore, despite Hao and Wickerhauser's suggestion to the contrary, the technique is not amenable to scale up for the treatment of the volumes of starting material handled by commercial manufacturers (typically several batches per week of up to 10,000 1 per batch of Cohn Fraction V supernatant). Most importantly this process has not been able to reduce the levels of bound contaminating LPS to levels acceptable for clinical use.
Other prior processes for purifying AAG have not been successful in depleting LPS from AAG preparations to levels which are acceptable for clinical use. One such method involves adsorption and elution of AAG preparations from Detoxigel resins (Boutten et al Eur. J. Immunol. 22, 2687-2695 (1992)). The purpose of this method was to ensure LPS was depleted from an AAG preparation for use in in vitro studies examining the effects of added LPS on cytokine production. This chromatography medium is not however suitable for use in preparing products for human administration, and in any event, LPS levels were only reduced to 200 pg/mg (approx. 2 EU/mg) of protein (EU=endotoxin units). This level is still too high for products intended for human use, particularly at the AAG doses likely to be required clinically (for example from 10 g to 30 g per dose) e.g. in the treatment of drug toxicity.